Enzymatically hydrolyzed composition of skin rag and derivatives thereof

ABSTRACT

A process comprising (a) enzymatically hydrolyzing skin rag with a neutral or alkaline protease which is capable of decomposing collagen other than collagenase, and (b) N-acylating the resultant oligopeptide hydrolyzation products with a fatty acid type acylating agent having from 8-22 carbon atoms to form Nacylated oligopeptides useful as surfactants.

United States Patent Fujimoto et a1.

Aug. 5, 1975 ENZYMATICALLY HYDROLYZED COMPOSITION OF SKIN RAG AND DERIVATIVES THEREOF Inventors: Yasu Fujimoto, Yokohama;

Masayuki Teranishi; Ikuo Matsukuma, both of Tokyo, all of Japan Assignee: Kyowa I-Iakko Kogyo Kabushiki Kaisha, Tokyo, Japan Filed: Oct. 13, 1972 Appl. No.: 297,461

Foreign Application Priority Data Oct. 28, 1971 Japan 46-10985 US. Cl 195/29; 195/6; 260/112 R;

252/544; 424/359 Int. Cl. ClZB l/00 Field of Search 195/29, 6

[56] References Cited UNITED STATES PATENTS 2,857,317 10/1958 Grimm 195/6 3,034,852 5/1962 Nishihara... 195/6 X 3314,86] 4/1967 Fujii 195/6 3,530,037 9/1970 Nishihara 195/6 Primary ExaminerLione1 M. Shapiro Assistant ExaminerRobert J. Warden Attorney, Agent, or Firm-Phillips, Moore, Weissenberger, Lempio & Strabala [5 7] ABSTRACT 2 Claims, No Drawings ENZYMATICALLY HYDROLYZED COMPOSITION OF SKIN RAG AND DERIVATIVES THEREOF BRIEF SUMMARY OF INVENTION This invention relates to (a) a process for producing enzymatically hydrolyzed composition of skin rag by treating skin rag with a protease, (b) a process for producing Naeyl derivatives by subjecting the said enzymatically hydrolyzed composition to acylation reaction by using a long chain fatty acid-type acylating agent, and (c) the utilization of the said N-acyl derivatives as surfactant.

DETAILED DESCRIPTION OF INVENTION Generally, a large amount of skin rag (which denotes animal skin rag such as e.g. hide rag, leather rag and the like) is brought forth as waste at various leather factories. The unpleasant smell of the skin rag itself and the smell which is generated during the after-treatment of the rag have caused questions of air pollution and public health to arise and for this and other reasons, the rag has not yet been used effectively in spite of strong desire, for a long period of time.

The collagen fiber, which is part of the main composition of skin, has a helical structure. It is said that this collagen helix is stable to protease other than collagenase under normal conditions. For example, it has been reported that various enzymes, such as pepsin, trypsin, Promnase (commercial product available from Kaken Kagaku Kabushiki Kaisha, Japan), Proctase (commercial product available from Meiji Seika Kabushiki Kaisha, Japan) etc., digest only the peptide portion (less than 5% of the entire molecule) which projects from the helix structure at the terminal of a collagen molecule, but do not affect the helix structure itself [Kobunshi (high molecule), Vol.19, pp. 355-361 (1970)].

We have extensively studied the problem of the economical and effective utilization of skin rag, and have now made the discovery that skin rag is easily decomposed into low molecular oligopeptides by a certain type of neutral and/or alkaline protease. We have also discovered the interesting fact that such hydrolysis proceeds with a very slow speed to completion, i.e., amino acid monomers.

The hydrolysis of protein is classified into three types, i.e., acidic hydrolysis, alkaline hydrolysis and enzymatic hydrolysis, depending upon the hydrolyzing agent in use. The first two types of hydrolysis have the disadvantage that the peptides and amino acids produced are liable to be further decomposed into undesired fumins. Furthermore, when applied to the hydrolysis of proteins into low molecular peptides, which is a purpose of the present invention, the first two types of hydrolysis have the disadvantage that the hydrolysis proceeds to the production of amino acid monomers owing to the difficulty of controlling these hydrolyzation processes.

Conventional techniques of alkaline hydrolysis of skin rag under pressure have also the aforementioned disadvantages. Furthermore, enzymatic hydrolysis has been conventionally used either for the purpose of obtaining collagen with simultaneous removal of some proteins (such as albumin. globulin and viscouse protein) which are undesirable for the production of leather or for the purpose of producing high molecular proteins such as gelatin.

According to the process of the present invention, it is possible to obtain directly from skin rag, under mild conditions, a hydrolysate containing little amino acid monomer, by using a neutral or alkaline protease which is capable of decomposing collagen other than collagenase.

Skin rag, which may be used as raw material for the purpose of the present invention, includes any and all skin rag brought forth as waste in leather manufacturing. Usually, either raw or tanned skin rag of cow, horse, goat, pig, sheep, etc., may be used for the process of the present invention.

The hydrolysis is carried out by immersing skin rag in water, for example, by soaking or suspending. In this case, the amount of the water is preferably about 4-50 times of that of the skin rag to be immersed, although it is not necessary to specifically limit the amount. It is difficult to carry out an enzymatic action on skin rag without pretreatment because of its intricate structure. Thus, it is sometimes necessary to denature the substrate prior to decomposition. It is possible to improve the degree of decomposition when the skin rag used in the present invention is denatured by heating. For example, the skin rag may be suspended or soaked in water and then subjected to heat treatment prior to enzymatic decomposition. It is generally preferable to heat the skin rag at a temperature of about C to about C for about 0.5 to about 5 hours. When the skin rag is subjected to such heat treatment, the degree of decomposition may be further improved by discarding the aqueous solution used for the heat treatment and carrying out the enzymatic decomposition in fresh water in which the skin rag is soaked or suspended.

Protease, which may be used for the hydrolysis, may be of animal, plant or microorganic origin. Although either a neutral or an alkaline protease which is capable of decomposing collagen other than collagenase may be used, better result may be obtained by using an alkaline protease which is capable of decomposing collagen. It is also possible to use various types of enzyme in combination. Typical enzymes employable are exemplified by protease of hay bacillus, Epolozyme (commercial product available from Kyowa Hakko Kogyo K. K., Japan), Tacynase (commercial product available from Hakko Kogyo K. K., Japan), or Prozyme (commercial product available from Amano Seiyaku K. K., Japan).

The conditions of the hydrolysis, of course, depend upon the enzyme employed, and the important factors are the pH and temperature of the reaction mixture. In all events, the pH and temperature should be determined so that the activity of the enzyme employed may be effectively utilized. The pH may be adjusted to a suitable value by a suitable alkaline or acidic substance, such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, hydrochloric acid, sulfuric acid, acetic acid, etc. It is also possible to use a suitable buffer solution.

The hydrolysis may be carried out in the following manner.

Immediately after immersing or suspending skin rag in water or after the said pretreatment, the reaction mixture is adjusted to optimum temperature (e.g. 2070C) and pH (e.g. 7-12), which may vary depending upon the types of enzyme which is added thereto. The amount of the enzyme to be added may vary, depending upon the activity, and is preferably within a range of about 0.1 to about 5.0% based on the weight of the substrate. The pH of the reaction mixture may be adjusted to optimum for the enzyme used in the above-mentioned manner during the reaction. lf dc sired, a suitable antiseptic may be added. The reaction can be carried out as long as the enzyme shows its activity and accelerates the decomposition (usually, for example, for 4-24 hours).

After the completion of the hydrolysis, the reaction mixture is, for example, heated from about 80 to about 100C to inactivate the enzyme and the insoluble matter is removed therefrom. The solution is then concentrated or dried in conventional manner, to give the powder. It is usually preferable to carry out the concentration and drying at a temperature below about 60C to avoid over-heating. The resultant hydrolysate is sometimes colored because the coloring matters which elute from the skin rag during the decomposition adhere to the hydrolysate. If desired, a further refined hydrolysate may be obtained by the following treatment:

A hydrolysate solution, from which the insoluble matter is removed, is heated at a temperature below about 60C, simultaneously with treatment with active carbon in an amount of about 1 to about weight based on the amount of the solids in the hydrolysate. It is also possible to use a suitable decoloring resin in place of the active carbon. The coloring matters may be removed significantly by this treatment. When a still further purification is required, or when fine powders of some types of active carbon contaminate the hydrolyzed solution, coloring matters, inorganic salts, active carbon and a part of peptides may be precipitated either by concentrating the solution to one-tenth and adding the same to about 10 times this amount of methanol (by volume) or by adding the said methanol to the said concentrated solution. After removing the precipitates, a colorless and transparent solution of peptides is obtained. The solution may be dried in conventional manner to give a white or slightly colored powder.

According to the present invention, a hydrolysis is easily achieved with a yield of about 40-50% by only a single hydrolyzing treatment. The reaction residue may be subjected to further hydrolyzing reactions to obtain a hydrolysis yield of about 85-95% by repeating.

The hydrolyzed composition, obtained by the process of the present invention, contains only a small amount of amino acid monomers and represents a mixture of various peptides. The amino acid components of this mixture are rich in glycine, proline and hydroxyproline, but very poor in sulfur-containing amino acids such as cystine, cysteine, etc. Thus, it is possible to not only protect human skin by using this hydrolysate as raw material, for example, for cosmetics and the like, which are placed in direct contact with human body, but also to inhibit deleterious influences upon the human body by using this hydrolysate for various materials which may directly or indirectly be placed in contact with human body.

The present invention further provides a process for producing N-acyl derivatives of the enzymatically hydrolyzed composition of skin rag obtained in the manner stated hereinbefore by subjecting the composition to a condensation reaction with a long chain fatty acidtype acylating agent, and the invention further provides for the utilization of the derivatives as surfactants which are usable as additives to detergents, cosmetics and the like, as fully and clearly described hereinafter.

Generally, the polymerization degree of peptides is important when an acylated product of peptide is used as a surfactant, because it is closely related to the stability of the final product to hard water and its efficiency as a surfactant. Peptides having a high polymerization degree, exhibit excellent stability to hard water and/or acid, but exhibit the liability of the reduction of efficiency as surfactants. Also the existence of amino acid monomer is not preferable with respect to skin permeability. It is preferable to use peptides having a polymerization degree of about 2 to about 7, which have desirable balance as surfactants and desirable properties.

The hydrolyzed composition obtained by the present invention, comprises mainly peptides having a polymerization degree of about 4 to about 7 and contains little amino acid monomers. The N-acylated product of these peptides has properties of good wettability, diffusing, homogenizing, washing, and surfactant, which is advantageous for less stimulating cosmetics.

It is possible to use any type of known fatty acid-type acylating agent having 8-22 carbon atoms as the acylating agent of the present invention; however, it is preferable to use fatty acid halides (especially chlorides) or fatty acid anhydrides. As the fatty acid, it is possible to use caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, palmitoleic acid, linoleic acid, or a mixture thereof, for example, beef tallow fatty acid, beef tallow hardened fatty acid, whale oil hardened fatty acid, beef stearic acid, beef tallow distilled fatty acid, coconut oil fatty acid, coconut oil decolorized fatty acid, rice bran distilled fatty acid and so on. Especially good result can be obtained by using acid chloride of these fatty acids.

The amount of the acylating agent may vary, depending upon the use of the acylated product as surfactant. It is most preferable to use the agent in the equivalence to the terminal amino acid group or not exceeding the equivalence, but the amount may be more than equivalence. However, the use of acylating agent in excessive amouht is not preferable because it may cause the introduction of large amounts of unreacted acid chloride and hydrolyzed fatty acid.

The condensation reaction of a hydrolyzed composition with an acylating agent may be carried out by dissolving the hydrolyzed composition in water and adding the acylating agent thereto with a vigorous agitation in a conventional manner, simultaneously with dropping alkali to keep the reaction system in an alkaline state. It is desirable to drop the acylating agent gradually, avoiding rapid dropping. lt is preferable to keep the reaction system in an alkaline state during the dropping. However, an excessively alkaline state accelerates the hydrolysis of acid chloride, therefore, it is most preferable to keep an alkaline state of about pH 9.0 to about pH 12.5.

The reaction is completed by heating the reaction mixture to about 50 to 60C, and yields N-aeyl derivatives of the hydrolyzed composition. As the reaction mixture contains unreacted fatty acid chloride, hydrolyzed fatty acid and various salts, oily material rises to the surface when the reaction mixture is acidified with a suitable acid (e.g. sulfuric acid) after the completion of the reaction. To this mixture an organic solvent in a suitable amount (conveniently 0.5 'to' 2 times by vol ume) is added with shaking, and allowed to stand until the mixture separates into three layers, i.e. water, oil

and organic solvent. As the organic solvent, it is possible to use any organic solvent which is inactive to the product. barely soluble or insoluble in water and capable of dissolving fatty acid. However, certain organic solvents cause emulsification and make it 'difficultto separate the mixture into three layers. As the solvent for good separation, it is preferable to use halogenized hydrocarbons (chloroform, carbon tetrachloride, ethylene dichloride, tetrachloroethane, trichloroethylcne, perchloroethylene, vetc.). The organic solvent layer contains fatty acid and its chloride; while the aqueous layer contains salts. After removing the organic solvent and aqueous layers, the oil layer is adjusted to a neutral or an alkaline state to give the alkali salt of the N-acyl derivative.

As the product obtained by the present invention is a watersoluble oil having an adhering property and excellent qualities of solubilizing, emulsifying, dispersing and foaming, it may be used for various purposes as a surfactant, for example, dispersing agent, emulsifying agent. textile treating agent, solubilizing agent and foaming agent. The present acylated derivative consists of peptide compounds and is therefore substantially harmless to the human body. so that it may be advantageously added to cosmetics, home detergents and the like.

The process of the present invention is further described in the following examples. In place of the ani mal skin rag used in the examples, both raw and tanned hide rag and leather rag of various other animals may be used in a similar manner. It is not necessary to limit the size of skin rag, and a large piece of skin may be used, though it is advantageous to cut it into a suitable size for shortening the time of hydrolysis. Also, it is possible to carry out N-acylation by using the abovementioned various fatty acid chlorides.

Hitherto only collagenase has been known as enzyme which is capable of decomposing collagen. It is now possible according to the present invention to provide other enzymes than collagenase which are capable of decomposing collagen by treating skin rag with a neutral or alkaline protease specified hereinbefore.

EXAMPLE 1 Chrome-tanned leather rag of cow (400 g) was suspended in water (8 1) and heated in boiling water for 1 hour. After cooling, the resultant mixture was adjusted to a pH 7.5 with 2N caustic soda and heated up to 45C. To the mixture Tacynase (2 g) (commercial product available from Kyowa Hakko Kogyo K.K., Japan) was added, and the reaction was carried out for hours. After the completion of the reaction, the reaction mixture was heated at 80C for 15 minutes to inactivate the enzyme and was then weakly acidified with 2N HCl. The insoluble matter was removed from the reaction solution to give a filtrate which was then concentrated to one-third volume at a temperature below 60C under reduced pressure, and was freeze-dried to give 158 g of slightly colored powder. The product was highly hygroscopic and easily soluble in water.

EXAMPLE 2 A mixture of skin rags of cow and pig (500 g) was suspended in water 10 l and heated in a boiling water for 2 hours. After cooling, the reection mixture was adjusted to a pH 10.0 with 2N caustic soda and heated up to 47C. To the mixture Epolozymc (2.5 g) (commercial product available from Kyowa Hakko Kogyo K.K., Japan) was added, followed by reaction for 24 hours. After the completion of the reaction, the mixture was heated at C for 30 minutes to inactivate the enzyme and was weakly acidified with 2N HCl. The insoluble matter was removed from the reaction mixture to give a filtrate which was treated with active carbon (5% based on the filtrate) at 5060C. After the carbon treatment, the filtrate was concentrated to one-tenth volume and the concentrate was added to about 10 times by volume of methanol with agitation. After removing the precipitate, a colorless and transparent solution was concentrated at a temperature below 60C under reduced pressure to evaporate the methanol almost completely, to which fresh water was added and freeze-dried to give 183 g of almost white powder. The product was barely colored as compared with the product obtained in Example 1. The average molecular weight measured by analyzing the terminal group of nitrogen was about 640. The composition of the product obtained is as follows:

EXAMPLE 3 The enzymatieally hydrolyzed composition obtained in a similar manner to that described in Example 2 g) was dissolved in water (300 ml) and the pH of the mixture was adjusted to 10.0 with 10% caustic soda. Lauroyl chloride (44g) was gradually dropped into the solution at 30C with vigorous agitation and with simultaneous adjustment of the pH to about 10.0 with 10% caustic soda. After dropping for 45 hours, the reaction mixture was heated at 50-55C for 1 hour to complete the reaction. The resultant reaction mixture was slightly white and turbid. The mixture was then adjusted to a pH of 3.0 with 30% sulfuric acid, whereupon creamy matter rose to the surface. An equal volume of ethylenedichloride was added to the mixture. After agitation, the mixture was allowed to stand to separate into three layers of water, oil and ethylenedichloride. The oil layer was separated and adjusted to a pH 8.0-9.0 with 25% caustic soda, whereupon a yellowish and transparent material having a strong adhering property was obtained. The product was powder of N- acylated product of the hydrolyzed composition which was easily soluble in water and had a good foaming property.

EXAMPLE 4 Process for the production of shaving cream:

(a) Nlauroyl compound of an enzymatieally hydrolyzed composition of skin rag of cow 10.0 (b) Stearic acid 10.0 (c) Mineral oil 2.0 (d) Butyl stearate 5.0 7: (e) Sperm head oil 5.0 (f) Antiseptic (methyl-p-benzoate) 0.1 71 (g) Perfume a small amount (h) Water 67.9 7:.

The above ingredients, except perfume, were admixed and boiled, followed by a cooling to a temperature below 35C with slow kneading. To the mixture, the perfume was added and well kneaded to give shaving cream.

EXAMPLE Process for the production of liquid shampoo:

(a) N-stearoyl compound of an enzymatically hydrolyzed composition of skin rag of cow 30.0 7: (b) Sodium arginate 0.9 "/1 (c) Propylene glycol 3.0 71 (d) Antiseptic (methyl-p-benzoate) 0.1 '7! (e) Perfume a suitable amount (f) Water 66.0 71

EXAMPLE 6 Process for the production of liquid cream:

N-acylated product of an enzymatically hydrolyzed composition of skin rag of pig by means of whale oil hardened fatty acid chloride 15.0 (b) Stearic acid 10.0 (c) Triethanol amine 4.0 70 (d) Barium hydroxide 2.0

Antiseptic (methyl-p-hcnzoatu) 0.

( l '22 Water 68.9 7:

According to the amounts stated above, water was added to a mixture of the acylated product, stearic acid Component (A) and component (B) were separately prepared in solution. Component (A) was heated to 85C and admixed with component (B) with vigorous agitation to produce a vanishing cream composition.

What is claimed is:

l. A process for preparing a skin rag hydrolysate containing mainly peptides having a polymerization degree of from about 4 to about 7, comprising treating skin rag with an alkaline protease, other than collagenasc, which protease is capable of decomposing collagen, at a pH of from about 7 to 12 and at a a temperature of from about 47 to C for a period of from about 4 to 24 hours, the amount of said protease being from about 0.1 to 0.5% based on the weight of said skin rag.

2. The process of claim 1, wherein the protein element of the skin rag is denatured prior to treatment with the protease.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3898129 DATED August: 5, 1975 |NV}ENTOR(S) YASUO FUJIMOTO et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

[75] Inventors: "Yasu Fujimoto" should read --Yasud Fujimoto-.

Signed and fiealed this twenty-eight Day of October 1975 A ttest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner 0[ Patents and Trademarks 

1. A PROCESS FOR PREPARING A SKIN RAG HYDROLYSATE CONTAINING MAINLY PEPTIDES HAVING A POLYMERIZATION DEGREE OF FROM ABOUT 4 TO ABOUT 7 COMPRISING TREATING SKIN RAG WITH AN ALKALINE PROTEASE OTHER THAN COLLAGENASE WHICH PROTEASE IS CAPABLE OF DECOMPOSING COLLAGEN AT A PH OF FROM ABOUT 7 TO 12 AND AT A TEMPERATURE OF FROM ABOUT 47* TO 70*C FOR A PERIOD OF FROM ABOUT 4 TO 24 HOURS HOURS THE AMOUNT OF SAID PROTEASE BEING FROM ABOUT 0.1 TO 0.5% BASED O THE WEIGHT OF SAID SKIN RAG..
 2. The process of claim 1, wherein the protein element of the skin rag is denatured prior to treatment with the protease. 